Restoration of the responsiveness of purified guanylate cyclase to nitrosoguanidine, nitric oxide, and related activators by heme and hemeproteins. Evidence for involvement of the paramagnetic nitrosyl-heme complex in enzyme activation.

Purification of soluble guanylate cyclase activity from rat liver resulted in loss of enzyme responsiveness to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitroprusside, nitrite, and NO. Responses were restored by addition of heat-treated hepatic supernatant fraction, implying a requirement for heat-stable soluble factor(s) in the optimal expression of the actions of the activators. Addition of free hematin, hemoglobin, methemoglobin, active or heat-inactivated catalase partially restores responsiveness of purified guanylate cyclase to MNNG, NO, nitrite, and nitroprusside. These responses were markedly potentiated by the presence of an appropriate concentration of reducing agent (dithiothreitol, ascorbate, cysteine, or glutathione), which maintains heme iron in the ferro form and favors formation of paramagnetic nitrosyl . heme complexes from the activators. High concentrations of heme or reducing agents were inhibitory, and heme was not required for the expression of the stimulatory effects of Mn2+ or Mg2+ on purified guanylate cyclase. Preformed nitrosyl hemoglobin (10 micron) increased activity of the purified enzyme 10- to 20-fold over basal with Mn2+ as the metal cofactor and 90- to 100-fold with Mg2+. Purified guanylate cyclase was more sensitive to preformed NO-hemoglobin (minimally effective concentration, 0.1 micron) than to MNNG (1 micron), nitroprusside (50 micron), or nitrite (1 mM). A reducing agent was not required for optimal stimulation of guanylate cyclase by NO-hemoglobin. Maximal NO-hemoglobin-responsive guanylate cyclase was not further increased by subsequent addition of NO, MNNG, nitrite, or nitroprusside. Activation by each agent resulted in analogous alterations in the Mn2+ and Mg2+ requirements of enzyme activity, and responses were inhibited by the thiol-blocking agents N-ethylmaleimide, arsenite, or iodoacetamide. The results suggest that NO-hemoglobin, MNNG, NO, nitrite, and nitroprusside activate guanylate cyclase through similar mechanisms. The stimulatory effects of preformed NO-hemoglobin combined with the clear requirements for heme plus a reducing agent in the optimal expression of the actions of MNNG, NO, and related agents are consistent with a role for the paramagnetic nitrosyl . heme complex in the activation of guanylate cyclase.     

Activation of particulate guanylate cyclase by nitroprusside and MNNG after filipin treatment.

Particulate guanylate cyclase from rat lung was stimulated less than 2-fold by agents capable of activating the soluble guanylate cyclase, including sodium nitroprusside, MNNG, azide and hydroxylamine. The action of the first two agents was potentiated by 10 mM 2-mercaptoethanol, and that of the last two by catalase. Pretreatment of the particulate enzyme with the polyene antibiotic, filipin, potentiated the stimulatory effects of the activators, activity with 1 mM nitroprusside in the presence of 2-mercaptoethanol being increased 10.4-fold over basal. The enzyme treated with filipin and nitroprusside showed less specificity for Mn2+, as it was able to use Mg2+ as sole cation more efficiently than the untreated enzyme. Since filipin is known to alter membrane fluidity by interacting with membrane cholesterol, it is proposed that the activity of membrane bound guanylate cylase may be regulated in part by the fluid state of the phospholipid matrix.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.     

Properties of the guanylate cyclase-guanosine 3':5'-monophosphate system of rat renal cortex. Activation of guanylate cyclase and calcium-independent modulation of tissue guanosine 3':5'-monophosphate by sodium azide.

The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism.     

Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca²⁺-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca²⁺. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn²⁺-dependent and decreased 85% when either Mg²⁺ or Ca²⁺ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn²⁺ or Mg²⁺, but not with Ca²⁺, as sole divalent cation. Improved utilization of Mg²⁺ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg²⁺ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg²⁺ or Mn²⁺ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca²⁺. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.     

Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy     

Under anaerobic conditions, soluble guanylate cyclase is specifically stimulated by glutathione

Various thiols exert non-specific effects on the activity of soluble guanylate cyclase under aerobic conditions. We studied the effects of thiols under anaerobic conditions (pO2 less than 6 Torr) on soluble guanylate cyclase, purified from bovine lung. Reduced glutathione stimulated the enzyme concentration-dependently with half-maximal enzyme stimulation at a concentration of about 0.5 mM. The extend of maximal enzyme stimulation (up to 80-fold) was comparable with the activation by NO-containing substances. The activation by glutathione was additive with the effect of sodium nitroprusside. Cysteine and various other thiols increased the enzyme activity 20-fold and 2- to 5-fold, respectively. The stimulatory effect of these thiols was not related to their reducing potency. Activation of soluble guanylate cyclase by glutathione was dose-dependently reduced in the presence of other thiols (cysteine greater than oxidized glutathione greater than S-methyl glutathione). Under aerobic conditions or with Mn-GTP as substrate, the effect of glutathione on soluble guanylate cyclase was suppressed. The results suggest a specific role for glutathione in the regulation of soluble guanylate cyclase activity and a modulation of this effect by redox reactions and other intracellular thiols.     

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.     

Appearance of magnesium guanylate cyclase activity in rat liver with sodium azide activation.

Native soluble and particulate guanylate cyclase from several rat tissues preferred Mn2+ to Mg2+ as the sole cation cofactor. Wtih 4mM cation, activities with Mg2+ were less than 25% of the activities with Mn2+. The 1 mM NaN3 markedly increased the activity of soluble and particulate preparations from rat liver. Wtih NaN3 activation guanylate cyclase activities wite similar with Mn2+ and Mg2+. Co2+ was partially effective as a cofactor in the presence of NaN3, while Ca2+ was a poor cation with or without NaN3. Activities with Ba, Cu2+, or Zn2+ were not detectable without or with 1 mM NaN3. With soluble liver enzyme both manganese and magnesium activities were dependent upon excess Mn2+ or Mg2+ at a fixed MnGTP or MgGTP concentration of 0.4 mm; apparent Km values for excess Mn2+ and Mg2+ were 0.3 and 0.24 mM, respectively. After NaN3 activation, the activity was less dependent upon free Mn2+ and retained its dependence for free Mg2+, at 0.4 mM MgGTP the apparent Km for excess Mg2+ was 0.3 mM. The activity of soluble liver guanylate cyclase assayed with Mn2+ or Mg2+ was increased with Ca2+. After NaN3 activiation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+. After NaN activation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+ or Mg2+. The stimulatory effect of NaN2 on Mn2+-and Mg2+-dependent guanylate cyclase activity from liver or cerebral cortex supernatant fractions required the presence of the sodium azide-activator factor. With partially purified soluble liver guanylate cyclase and azide-activator factor, the concentration (1 mjM) of NaN3 that gave half-maximal activation with Mn2+ or Mg2+ was imilar. Thus, under some conditions guanylate cyclase can effectively use Mg2+ as a sole cation cofactor.     

Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.     

Activation of guanylate cyclase from rat liver and other tissues by sodium azide.

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.     

Adenine nucleotide regulation of particulate guanylate cyclase from rat lung.

Adenine nucleotides activate basal particulate guanylate cyclase in rat lung membranes. Activation is specific for adenine and not guanine, cytidine or uridine nucleotides. The concentration of adenine nucleotides yielding half-maximum activation of particulate guanylate cyclase is 0.1 mM and this nucleotide activates the enzyme by increasing maximum velocity 11-fold without altering affinity for substrate. Activation is specific for particulate guanylate cyclase, since soluble enzyme is inhibited by adenine nucleotides. Similarly, activation is specific for magnesium as the enzyme substrate cation cofactor, since adenine nucleotides inhibit particulate guanylate cyclase when manganese is used. Adenine nucleotide regulation of particulate guanylate cyclase may occur by a different molecular mechanism compared to other activators, since the effects of these nucleotides are synergistic with those of detergent, hemin and atrial natriuretic peptides. Cystamine inhibits adenine nucleotide activation of particulate guanylate cyclase at concentrations having minimal effects on basal enzyme activity suggesting a role for critical sulfhydryls in mechanisms underlying nucleotide regulation of particulate guanylate cyclase. Purification and quantitative recovery of particulate guanylate cyclase by substrate affinity chromatography results in the loss of adenine nucleotide regulation. These data suggest that adenine nucleotides may be important in the regulation of basal and activated particulate guanylate cyclase and may be mediated by an adenine nucleotide-binding protein which is separate from that enzyme.     

Activation of soluble splenic cell guanylate cyclase by prostaglandin endoperoxides and fatty acid hydroperoxides.

Purified prostaglandin endoperoxides (PGG2 and PGH2) and hydroperoxides (15-OOH-PGE2) as well as fatty acid hydroperoxides (12-OOH-20:4, 15-00H-20:4, and 13-OOH-18:2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures described (in the miniprint supplement) for the preparation, purification, and characterization of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5- to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed half-maximal stimulation was achieved in the range of 2 to 3 micrometer. However, by extending the incubation time to 90 min similar maximal increases in specific activity could be achieved with 3 or 10 micrometer PGG2 or PGH2. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or glutathione (10 to 15 mM). Na2S2O4, not known as an effective reducing agent of disulfides, prevented but was relatively ineffective in reversing activation after it had been induced by PGG2. Pretreatment of the enzyme preparation with increasing concentrations of N-ethylmaleimide in the range of 0.01 to 1.0 mM prevented activation by PGG2 without affecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast PGE2, PGF2a, hydroxy fatty acids (13-OH-18:2, 12-OH-20:4) as well as saturated (18:0) monoenoic (18:1), dienoic (18:2), and tetraenoic (20:4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 micrometer. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG2 but not 15-OOH-PGE2 (the major buffer-rearrangement product of PGG2) is most likely an activator. In the case of PGH2, a rapidly generated (30 s) metabolite of PGH2 was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH2 as an apparent activator of the enzyme. The combined effects of PGG2 and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryl groups that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.     

Properties and subcellular distribution of guanylate cyclase activity in rat renal medulla: correlation with tissue content of guanosine 3',5'-monophosphate.

The properties of the guanylate cyclase systems of outer and inner medulla of rat kidney were examined and compared with those of the renal cortex. A gradation in steady-state cyclic guanosine 3',5'-monophosphate (cGMP) levels was observed in incubated slices of these tissues (inner medula greater than outer medulla greater than cortex). This correlated with the proportion of total guanyl cyclase activity in the 100 000 g particulate fraction of each tissue, but was discordant with the relative activities of guanylate cyclase (highest in cortex) and of cGMP-phosphodiesterase (lowest in cortex) in whole tissue homogenates. Soluble guanylate cyclase of cortex and inner medulla exhibited typical Michaelis-Menten kinetics with an apparent Km for MnGTP of 0.11 mM, while the particulate enzyme from inner medulla exhibited apparent positive cooperative behavior and a decreased dependence on Mn2+. Thus, the particulate enzyme could play a key role in regulating cGMP levels inthe intact cell where Mn2+ concentrations are low. The soluble and particulate enzymes from inner medulla were further distinguished by their responses to several test agents. The soluble enzyme was activated by Ca2+, NaN3, NaNo2 and phenylhydrazine, whereas particulate activity was inhibited by Ca2+ and was unresponsive to the latter agents. In the presence of NaNo2, Mn2+ requirement of the soluble enzyme was reduced and equivalent to that of the particulate preparation. Moreover, relative responsiveness of the sollble enzyme to NaNO2 was potentiated when Mg2+ replaced Mn2+ as the sole divalent cation. These changes in metal requirements may be involved in the action of NaNO2 to increase cGMP in intact kidney. Soluble guanylate cyclase of cortex was clearly more responsive to stimulation by NaN3, Nano2, and phenylhydrazine that was soluble activity from either medullary tissue. The effectiveness of the agonists on soluble activity from outer and inner medulla cound also be distinguished. Accordingly, regulation and properties of soluble guanylate cyclase, as well as subcellular enzyme distribution, and distinct in the three regions of the kidney.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.     

Soluble guanylate cyclase in molecular mechanisms underlying the therapeutic action of drugs

The influence of ambroxol (a mucolytic agent) on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside (SNP) and Sin-1) were investigated. Ambroxol in the range of concentrations from 0.1 to 10 ??M had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the SNP-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC₅₀ values of 3.9 and 2.1 ??M, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin (an antimalarial agent) on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1?100 ??M) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the SNP-induced activation of human platelet guanylate cyclase with the IC₅₀ value of 5.6 ??M. Artemisinin (10 ??M) also inhibited (by 71 ± 4.0%) the activation of the enzyme by a thiol-dependent NO-donor, the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 ??M), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the signaling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.     

The involvement of catalytic site thiol groups in the activation of soluble guanylate cyclase by sodium nitroprusside

Sodium nitroprusside, a potent activator of soluble guanylate cyclase, potentiated mixed disulfide formation between cystine, a potent inhibitor of the cyclase, and enzyme purified from rat lung. Incubation of soluble guanylate cyclase with nitroprusside and [35S]cystine resulted in a twofold increase in protein-bound radioactivity compared to incubations in the absence of nitroprusside. Purified enzyme preincubated with nitroprusside and then gel filtered (activated enzyme) was activated 10- to 20-fold compared to guanylate cyclase preincubated in the absence of nitroprusside and similarly processed (nonactivated enzyme). This activation was completely reversed by subsequent incubation at 37 degrees C (activation-reversed enzyme). Incorporation of [35S]cystine into guanylate cyclase was increased twofold with activated enzyme, while no difference was observed with activation-reversed enzyme, compared to nonactivated enzyme. Cystine decreased the activity of nonactivated and activation-reversed enzyme about 40% while it completely inhibited activated guanylate cyclase. Mg+2- or Mn+2-GTP inhibited the incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. Also, diamide, a potent thiol oxidant that converts juxtaposed sulfhydryls to disulfides, completely blocked incorporation of [35S]cystine into nonactivated or activated guanylate cyclase. These data indicate that activation of soluble guanylate cyclase by nitroprusside results in an increased availability of protein sulfhydryl groups for mixed disulfide formation with cystine. Protection against mixed disulfide formation with diamide or substrate suggests that these groups exist as two or more juxtaposed sulfhydryl groups at the active site or a site on the enzyme that regulates catalytic activity. Differential inhibition by mixed disulfide formation of nonactivated and activated enzyme suggests a mechanism for amplification of the on-off signal for soluble guanylate cyclase within cells.     

Activation of mouse splenic lymphocyte guanylate cyclase by calcium ion.

The guanosine 3',5'-cyclic monophosphate (cGMP) level in the mouse splenic lymphocytes was increased about 2- to 3-fold by concanavalin A. This increase was completely dependent on the presence of Ca2+ in the medium. Homogenates of mouse splenic lymphocytes contained significant guanylate cyclase [EC 4.6.1.2] activity in both the 105,000 X g (60 min) particulate and supernatant fractions and both fractions required Mn2+ for full activity. Calcium ion (3mM) activated soluble guanylate cyclase 3-fold at a relatively low concentration of Mn2+ (less than 1mM) but inhibited the particulate enzyme slightly at all Mn2+ concentrations tested. Concanavalin A itself did not stimulate either fraction of guanylate cyclase. Thus these results suggest that elevation of the cGMP level in lymphocytes by concanavalin A might be brought about by stimulation of Ca2+ uptake and activation of soluble guanylate cyclase by the latter.